Bile salt hydrolase in non-enterotoxigenic Bacteroides potentiates colorectal cancer

Bile salt hydrolase (BSH) in Bacteroides is considered a potential drug target for obesity-related metabolic diseases, but its involvement in colon tumorigenesis has not been explored. BSH-expressing Bacteroides is found at high abundance in the stools of colorectal cancer (CRC) patients with overweight and in the feces of a high-fat diet (HFD)-induced CRC mouse model. Colonization of B. fragilis 638R, a strain with low BSH activity, overexpressing a recombinant bsh gene from B. fragilis NCTC9343 strain, results in increased unconjugated bile acids in the colon and accelerated progression of CRC under HFD treatment. In the presence of high BSH activity, the resultant elevation of unconjugated deoxycholic acid and lithocholic acid activates the G-protein-coupled bile acid receptor, resulting in increased β-catenin-regulated chemokine (C-C motif) ligand 28 (CCL28) expression in colon tumors. Activation of the β-catenin/CCL28 axis leads to elevated intra-tumoral immunosuppressive CD25+FOXP3+ Treg cells. Blockade of the β-catenin/CCL28 axis releases the immunosuppression to enhance the intra-tumoral anti-tumor response, which decreases CRC progression under HFD treatment. Pharmacological inhibition of BSH reduces HFD-accelerated CRC progression, coincident with suppression of the β-catenin/CCL28 pathway. These findings provide insights into the pro-carcinogenetic role of Bacteroides in obesity-related CRC progression and characterize BSH as a potential target for CRC prevention and treatment.


March 2021
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Sample size 3. The tumor sizes were further analyzed by a ProgRes CapturePro version 2.10.0.1. 4. Signal intensity of IHC staining was measured on Fiji software (ImageJ version 1.53t). 5. For mRNA sequencing, gene expression quantification analysis was performed for all samples using STAR/RSEM tools. The following analysis was performed in NetworkAnalyst platform (https://www.networkanalyst.ca). 6. Flow cytometry data analysis was performed with Flow Jo Version 10 (BD Biosciences). 7. GraphPad Prism version 9.0 was used to analyze the statistics.
Bulk mRNA-seq output was listed in Supplementary Data 3 and the original sequencing data set has been deposited in GEO database under accession code GSE190298 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190298). Mouse and human shotgun metagenomics outputs were listed in Supplementary Data 1 and 2, and the sequencing datasets have been uploaded to the public database in National Library of Medicine under accession codes PRJNA786913 for mouse and PRJNA881471 for human. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA786913; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA881471. Source data are provided with this paper.
We collected stool samples from both male and female subjects. The information regarding the sex of each participant was collected by self-reporting via a questionnaire.
Participants were aged 28-86 years, Chinese, with a BMI of 18.07-33.06 kg/m2. Participants in CRC group were pathologically confirmed diagnosis of colorectal cancer without other combined tumors and distant metastasis. Participants in Control group had no no history of inflammatory bowel disease (Crohn's disease, ulcerative colitis), other malignant tumors or bowel resection. More detailed information related to health status (i.e., age, sex, BMI and biochemical indicators) were collected (Supplementary Table 1).
Healthy individuals and individuals who were diagnosed with CRC by colonoscopy and/or undergone colorectal surgery at the Peking University Third Hospital were enrolled. According to the cut-offs of BMI provided by the World Health Organization (WHO), individuals of BMI#25 kg/m2 belong to overweight group, and individuals of BMI<25 kg/m2 belong to lean group. Exclusion criteria are as follows: (1) Patients used antibiotics, probiotics preoperative four weeks; (2) Patients with a history of inflammatory bowel disease (Crohn's disease, ulcerative colitis); (3) Patients with a history of other malignant tumors; (4) Patients with a history of intestinal resection and (5) Patients receiving neoadjuvant therapy before surgery. Finally, 45 individuals (14 for control lean group; 11 for control overweight group; 11 for CRC lean group; 9 for CRC overweight group) were included in the study. All patients have signed informed consent forms prior to enrollment in the study. Participants can get their intestinal macrobiota composition data and fare compensation.
This study conformed to the ethical principles outlined by the Declaration of Helsinki and ethical approval for this study was obtained from the Ethics Committee of Peking University Third Hospital (IRB00006761-LM2022557).
There were at least 3 samples or replicates for each experiment in this manuscript. Especially for animal experiments, there were over 5 mice per group in order to obtain statistical significance. Sample size was estimated on the basis of sample availability and previous studies.

March 2021
Data All data representative of three or more independent experiments, as stated in the figure legends.
Human subjects were randomly selected. Healthy individuals and individuals who were diagnosed with CRC by colonoscopy and/or undergone colorectal surgery at the Peking University Third Hospital were enrolled as Control and CRC groups. According to the cut-offs of BMI provided by the World Health Organization (WHO), individuals of BMI#25 kg/m2 belong to overweight group, and individuals of BMI<25 kg/m2 belong to lean group. 6-8 week old mice were divided at random into experimental groups, with at least 5 mice per group, and the groups did not present differences in age or body weights before the treatments.
Investigators were blinded to group allocation during data collection and analysis. But during the treatment of live animals it was not blinded, as the treatment of each mouse would need to be known to the person handling the mice.

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In epidemiological studies, male sex has consistently shown strong associations with CRC incidence (PMID: 31631858). Male mice were applied in this study.
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Colons tumors were harvested and flushed with cold PBS. The collected colon tumors were further washed in cold colon buffer and cut into small fragments that were transferred into gentleMACS™ C Tubes with 10 mL pre-warm colon buffer containing 100 U/mL collagenase E. Cell dissociation was performed with gentleMACS™ Octo Dissociator. Isolated single cells (~1×106) were resuspended in FACS buffer and stained with LIVE/DEADTM fixable yellow dye and cell surface markers, including CD45, CD3e, CD4, CD8a, CD25. Before further staining with nuclear FOXP3, the cells were fixed and permeabilized with eBioscienceTM FOPX3/transcription factor staining buffer set.
Flow cytometry analysis were conducted by LSRFortessa SORP I (BD Biosciences).
Flow cytometry analysis were conducted by LSRFortessa SORP I. Flow cytometry data analysis was performed with Flow Jo Version 10.
No sorting was applied in this study.